The long-term goal of this project is to understand the mechanisms regulating proliferation and differentiation of spermatogonial stem cells in the human and monkey testis. In all primates two types of undifferentiated spermatogonia (Adark, Apale) exist and the differentiation of Apale spermatogonia into B spermatogonia is considered to be the initiating step in spermatogenesis in these species. Three Specific Aims will be addressed: Aim 1 will characterize the biology of human and monkey spermatogonial stem cells. In particular, the existence of clonogenic patterns of germ cell expansion and the impact of paracrine factors in adult testicular tissue will be investigated; Aim 2 will determine the signals responsible for the differentiation of Apale spermatogonia to B spermatogonia; and Aim 3 will determine the potential of xenografting to initiate or restore spermatogonial differentiation, meiotic progression and androgen production in human testicular tissue, in which spermatogenesis is compromised. These aims will be achieved using organ culture of human and monkey testicular tissue over periods of 72 hr and xenografts of testicular tissue into immunodeficient nude mice. These approaches allow us to expose limited amounts of primate testicular tissue to a variety of different conditions by modifying the in vitro conditions and applying various treatments to the graft recipients. We will use a newly developed approach to detect proliferating spermatogonial clones in whole mounts of testis tissue. The dual fluorescence labeling of bromodeoxyuridine and acrosin in conjunction with confocal microscopy allows the description of the clonogenic and spatial arrangement of proliferating spermatogonia at specific stages of the seminiferous epithelial cycle. Cross-sections of resin and paraffin-embedded cultured testicular fragments and dissected grafts will be labeled by the same approach. Quantitative morphometric analysis using both, the optical dissector and geometric approaches, is used to determine changes in spermatogonial cell types, numbers and proliferation indices. As yet the knowledge on the mechanisms regulating the initiation of spermatogonial self-renewal and differentiation is very limited. No well defined models for the kinetics of premeiotic germ cell development and the role of paracrine factors on spermatogonial proliferation and differentiation have been described for any species of primate. Therefore, the proposed studies of human and monkey should lead to fundamental advances in our understanding of the control of spermatogenesis in man. These new findings have clinical relevance as they may lead to new concepts in understanding and treatment of male infertility, developing new strategies for male contraception and exploring better and efficient strategies for preserving and restoring fertility in patients undergoing gonadotoxic oncological treatments.